The present invention relates to novel culturable cells with no nucleus, showing mitochondrial activity.
Amitochondrion has its own DNA (mitochondrial DNA: hereinafter, referred to also as “mtDNA”) and an autonomous genetic expressing system. Only 13 peptides, however, are encoded by mtDNA and all the other mitochondrial proteins including factors relating to the genetic expression system are encoded by nuclear genes. Therefore, it has been believed that mitochondria will not be maintained without nucleus. Conventionally, techniques for isolating and purifying mitochondria from liver, heart muscle, skeletal muscle, brain, platelets and so on have been established (cf. non-patent documents 1–3). Functions of mitochondria, such as enzyme activity, have been evaluated with purified mitochondria in various diseases including mitochondrial diseases, Parkinson's disease, Alzheimer's disease, and Huntington disease (cf. non-patent documents 3–6). However, isolated and purified mitochondria have no proliferation potency so that it is impossible to culture the mitochondria.
Platelets are unique cells in an organism. They separate from megakaryocytes and have mitochondria but no nucleus. However, platelets have no proliferation potency and their life lasts only several days in an organism. This has made it impossible to observe changes of mitochondria with passage of time.
On the other hand, mtDNA-less human cell lines, designated Rho0 cells, have been isolated (cf. non-patent document 7). Fusion of cells of a Rho0 cell line with enucleated cells having mutant mtDNA to effect cytoplasmic transfer has enabled culture of cells having the mutant mtDNA (cybrids) Cell lines, obtained by fusing Rho0 cells with enucleated cells from patients with a variety of mitochondrial diseases, have contributed to elucidation of mitochondrial disorders (cf. non-patent documents 8–10). However, the mitochondria of such a cybrid cell are always under the influence of the nucleus and there is the possibility that the change of mitochondria in cultured cells when a drug is administered to the cultured cells is attributable to an indirect action of the drug to the mitochondria through the nucleus. Accordingly, such cybrid cells have been unsatisfactory for the evaluation of direct action of a drug or the like to mitochondria. From those facts, it has been demanded to culture cells that have no nucleus and show mitochondrial activity governed by mitochondria.
<Non-Patent Document 1>
    Stumpf et al. Biochem Med 21, 182–189, 1979<Non-Patent Document 2>    Hatafi Y et al. A.B.B. 94 148, 1964. Methods in enzymology IV, 51–59, 1979<Non-Patent Document 3>    Haas R H, K Nakano et al. Ann Neurol 1995; 37: 714–722<Non-Patent Document 4>    Shoffner J M, Brown M D, et al. Genomics 17: 171–184, 1993<Non-Patent Document 5>    Grunewald T et al. Ann New York Academy Science 893: 203–213, 1999<Non-Patent Document 6>    Maurer I et al. Neurobiology of Aging 21: 455–62, 2000<Non-Patent Document 7>    GuM Cooper J M et al. Ann Neurol 44: 177–186, 1998<Non-Patent Document 8>    Chomyn A, et al. Proc Natl Acad Sci USA 89: 4221–4225, 1992<Non-Patent Document 9>    Dunbar D R, et al. Hum Mol Genet 5: 123–129, 1996<Non-Patent Document 10>    Chomyn A, et al. Am J Hum Genet 34: 966–974, 1994